The present invention relates to a method and kit for direct radiolabeling of a protein with a radiometal ion of a radionuclide that binds tightly to sulfhydryl groups, using one or more pendant sulfhydryl groups as endogenous ligands and optionally using an exogenous ligand which binds tightly to the radiometal ion to further stabilize the chelate.
It is known that certain radiometals bind tightly to sulfur ligands, including, e.g., Tc-99m from reduced pertechnetate, Re-186 and Re-188 ions, Cu-67 ions, Hg-197 ions and Bi-212 ions. Some of these radiometals have been bound to proteins, especially antibodies or antibody fragments, using conjugated chelating groups such as diethylenetriamine-pentaacetic acid (DTPA) or a variety of sulfur/nitrogen (S.sub.2 N.sub.2) chelators such as bis-thiosemicarbazones and the like.
Methods have been reported for binding Tc-99m ions directly to antibodies by pre-tinning the protein and then contacting the resultant material with pertechnetate. This procedure often does not work well and some of the radiometal is bound to sites which are comparatively labile in the presence of blood and other bodily fluids or tissues. The mechanism of the pre-tinning process is not well understood and the reasons for production of labile sites of labeling have not been elucidated.
It is also known that proteins containing disulfide bonds can be reduced to produce pendant sulfhydryl groups. If the disulfide bonds link polypeptide chains which are not themselves joined, e.g., antibody light/heavy chains, reductive cleavage thereof can dissociate the protein into smaller fragments. An example of this is the reductive cleavage of antibody F(ab').sub.2 fragments to form Fab' fragments using disulfide reducing agents such as cysteine, dithiothreitol, mercaptoethanol and the like.
Direct labeling of a protein has the advantage that chelate conjugates are obviated. However, attempts to use such radiolabeled proteins for in vivo applications, e.g., antibody-targeted tumor imaging and therapy, has revealed problems due to loss of the label to other organs which retain it and cause both high background for certain types of imaging and slow clearance of the label from organs such as the liver, spleen and kidneys. The latter problem can in turn cause hepatic and/or renal crisis for the therapy patient due to severe compromise of the affected organ by the high levels of radiation.
A need continues to exist for a direct method for radiolabeling a protein that produces a good yield of a stable radiolabeled product which retains the label in the presence of blood and other body fluids and tissues.